In the field of recombinant deoxyribonucleic acids (DNA) technology, plasmids are largely used to encode and express heterologous proteins of interest.
Recently, it has been shown that plasmid DNA may be useful for clinical applications, such as gene therapy and genetic immunization (Wolf et al., Science, 1990, 247, 1465-1468; WO-A-90/11092).
Plasmids are too large and complex to be produced in large quantities through synthetic means. Instead, plasmids must be produced in cells, and subsequently extracted, harvested and purified. Generally, plasmids are produced via bacterial fermentation and recovered by cell disruption. The persons skilled in the art know the fermentation techniques. Many of these techniques have been published and are routinely used (e.g. Sambrook et al., section 1.21, “Extraction and purification of plasmid DNA”, Molecular Cloning: a laboratory manual, second edition, cold Spring Harbor Laboratory Press, 1989). These methods involve the growth of the bacterial culture and replication of the plasmid, harvesting and lysis of the bacteria, isolation and purification of the plasmid DNA.
Accordingly, there is a need for large scale processes for extracting and recovering supercoiled plasmid DNA.
A large variety of cell disruption techniques is available, including mechanical or physical (e.g. high pressures, sonication, heat treatments), chemical (e.g. nonionic detergents, ionic detergents, organic solvents, alkali), or enzymatic (e.g. lysozyme) methods.
For pharmaceutical and immunization uses, plasmid DNA compositions must avoid the presence of impurities, such as genomic DNA, endotoxins and tRNA (transfer ribonucleic acids) or rRNA (ribosomal ribonucleic acids), and a decrease of yield due to a plasmid degradation and to the low-efficiency of the extraction method.
As supercoiled plasmid DNA is usually separated from both the larger host cell genomic DNA and from the smaller cellular RNAs and DNAs on the basis of their size, it is important to avoid shearing either the plasmid DNA or the genomic DNA. Shearing forces may damage genomic DNAs and produce genomic DNAs having the same size than plasmid DNAs. Shearing forces may also cut the plasmid DNAs and produce linearized plasmid DNAs. Linearized plasmid DNAs and genomic DNA fragments similar in size to the supercoiled plasmid DNAs may be particularly difficult to be separated from supercoiled plasmid DNAs.
For pharmaceutical and immunization uses, it is preferable to use supercoiled plasmid DNAs, which are smaller and more compact than relaxed closed circular plasmid DNAs and less vulnerable to enzymatic degradation. Cell disruption techniques may damage supercoiled plasmid DNAs and produce closed circular, linearized or fragmented plasmid DNAs, which increase the level of impurities and reduce the yield of supercoiled plasmid DNAs.
Several cell disruption techniques are commonly used to release intracellular product, particularly intracellular proteins.
Cell disruption techniques are classified into two main categories: the first one involves only physical forces to break the cells and the second one involves contact of chemical or enzymatic agents with the cells and destruction of the cell membrane, capsule or wall.
The physical forces involved in the first category may be:
high pressures or high temperatures (for example due to microfluidization, nebulization techniques or heat treatments),
cavitation (for example due to sonication techniques), impacts against solids (for example due to bead milling techniques) (e.g. see U.S. Pat. No. 6,455,287; Agerkvist I. et al., Biotechnol. Bioeng., 1990, 36, 1083-1089; U.S. Pat. No. 6,071,480).
The cell disruption depends on the conditions of residence time, pressure, temperature, agitation rate, shearing forces, impact forces . . . . These conditions are difficult to control and to monitor. Too weak physical forces may not break all the cells and lead to a non-optimal yield of plasmid DNAs. Too strong physical forces may damage free DNAs and lead to plasmid DNA fragmentation, create an unacceptable level of impurities and reduce the yield of supercoiled plasmid DNAs.
In the second category of cell disruption techniques, the enzymatic or chemical lysis of cells involves the mixing between the cell suspension and the lysing solution. This constitutes the technical field of the present invention. The mixing between the cell suspension and the lysing solution is a crucial step. Incomplete mixings, in particular due to a too short time of mixing, may result in an incomplete lysis of the cells, in a partial loss of the biological compound of interest, leading to non-optimal yield. On the contrary, a too long time of mixing may result in a too long contact between the cells and lysing solution, lead to the degradation of the biological compound of interest and of the genomic DNAs, produce genomic DNA fragments having the same size as the plasmid DNAs. Both situations will increase the costs of the biological final compound of interest. It is acknowledged in the prior art that to allow a full recovery of supercoiled plasmid DNAs, processing conditions must be very mild, particularly with respect to shearing forces during the mixing step.
U.S. Pat. No. 6,197,553 describes enzymatic lysis with lysozyme and heat treatment through a heat exchanger. This technique has the disadvantage of using 2 types of enzymes (lysozyme and RNase) and that the enzymes are from animal sources, which could lead to possible problems of safety due to contamination, among others by prions. Another disadvantage of this technique is that the enzymes must be subsequently discarded. This involves several purification steps, in particular gradient-based anion exchange chromatography and gradient-based reverse phase high performance liquid chromatography.
To automate the methods for purifying plasmid DNAs from cell, it has already been proposed to continuously pass the cell suspension to be lysed with a lysing solution through a static mixer (WO-A-00/05358). Usually, static mixers contain an internal helical structure which allows the cell suspension and the lysing solution to come into contact in an opposite rotary flow and force them to mix in a turbulent or laminar flow. Such mixers are described, for instance, in WO-A-00/05358 and U.S. Pat. No. 5,837,529. The technique using static mixer is difficult to scale-up due to the limits of internal diameter size and flow rates.
Others propose to continuously pass the cell suspension to be lysed and a lysing solution through a common tube (see U.S. Pat. No. 6,664,049). A major disadvantage of this technique is the great difficulty to scale-up due to the small size of the internal diameter of the tube. The solution described in this patent is not to scale-up the process but to multiply the tubes to increase the production. This solution requires a complex installation with multiple tubes and one or several pumps. This installation is difficult to control and to monitor, particularly to adjust the flow rates in each tube in order to obtain and to keep an efficient mixing.
There is a need for new scalable methods to prepare purified biological compounds of interest, in particular purified plasmid DNAs, and more particularly purified supercoiled plasmid DNAs.
These methods may have a high yield and produce biological compounds of interest with a good level of purity. It is also desirable to have scalable methods that can be used to produce large quantities of plasmid DNAs. These methods may also be fast, easy to use and easy for maintenance.
In addition, it is desirable to have flow-through methods to increase the robustness, reproducibility and ease of scale-up. It is desirable to have methods in two steps, the mixing step and the contact step, in order to control and to monitor them separately. It is desirable to have methods that can produce plasmid DNAs at a low cost. It is also desirable to prepare purified plasmid DNAs, without toxic chemicals, impurities, endotoxins or other compounds that would be prejudicial for their safety, efficacy or purity.
One of the objectives of the invention is to provide a flow-through and scalable method of mixing at least two fluids to allow chemical lysis of the cells suspended in one of these fluids.
A second objective of the invention is to propose a mixing device for such method.
A third objective of the invention is to provide a flow-through and scalable method for preparing biological compounds of interest, in particular supercoiled plasmid DNAs, using such a mixing device.
The present invention meets these needs and reaches these objectives.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.